PCSK9 acts as a key regulator of Aβ clearance across the blood–brain barrier

Despite the neurodegenerative disorder Alzheimer’s disease (AD) is the most common form of dementia in late adult life, there is currently no therapy available to prevent the onset or slow down the progression of AD. The progressive cognitive decline in AD correlates with a successive accumulation of cerebral amyloid-β (Aβ) due to impaired clearance mechanisms. A significant percentage is removed by low-density lipoprotein receptor-related protein 1 (LRP1)-mediated transport across the blood–brain barrier (BBB) into the periphery. Circulating proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to members of the low-density lipoprotein receptor protein family at the cell surface and targets them for lysosomal degradation, which reduces the number of functional receptors. However, the adverse impact of PCSK9 on LRP1-mediated brain Aβ clearance remains elusive. By using an established BBB model, we identified reduced LRP1-mediated brain-to-blood Aβ clearance due to PCSK9 across different endothelial monolayer in vitro. Consequently, the repetitive application of FDA-approved monoclonal anti-PCSK9 antibodies into 5xFAD mice decreased the cerebral Aβ burden across variants and aggregation state, which was not reproducible in brain endothelial-specific LRP1−/− 5xFAD mice. The peripheral PCSK9 inhibition reduced Aβ pathology in prefrontal cortex and hippocampus–brain areas critically involved in memory processing—and prevented disease-related impairment in hippocampus-dependent memory formation. Our data suggest that peripheral inhibition of PCSK9 by already available therapeutic antibodies may be a novel and easily applicable potential AD treatment

In vitro and in vivo testing of stereolithography (SLA)-manufactured haemocompatible photopolymers for blood pump

A major medical problem of state-of-the-art heart ventricular assist devices (LVADs) is device-induced thrombus formation due to inadequate blood-flow dynamics generated by the blood pump rotor. The latter is a highly complex device, with difficulties during conventional manufacturing through milling or casting. Therefore, the additive manufacturing technology relying on stereo-lithography (SLA) capable of producing parts of significantly increased freedom for a blood-flow-compatible, thrombus-risk-free design was chosen as novel and flexible technology for that type of application. However, as yet state-of-the-art SLA is not suitable to produce fully safe blood-contacting devices. Therefore, the present experiment covered chemical, mechanical, rheological, tribological, and complex biocompatibility characterization in accordance with i.a. ISO 10993 standards, including hemolysis and an acute thrombogenicity blood test on fresh animal blood (both as innovative laboratory tests to avoid animal usage in preclinical studies) with a special focus on testing demonstrators of miniaturized blood pump rotors. The conducted tests indicated acceptable biocompatibility of the material and a slight improvement in biocompatibility with surface modification. Additionally, a high biocompatibility of the tested materials was confirmed. Based on studies and simulations, stereolithography (SLA) as an additive manufacturing technology with significantly increased freedom for a blood-flow-compatible, thrombus-risk-free design was chosen as a novel and flexible technology basis in the 4DbloodROT project to enable future manufacturing of rotors with exceptional biomimetic complexity

The solute carrier SLC7A1 may act as a protein transporter at the blood-brain barrier

Despite extensive research, targeted delivery of substances to the brain still poses a great challenge due to the selectivity of the blood-brain barrier (BBB). Most molecules require either carrier- or receptor-mediated transport systems to reach the central nervous system (CNS). These transport systems form attractive routes for the delivery of therapeutics into the CNS, yet the number of known brain endothelium-enriched receptors allowing the transport of large molecules into the brain is scarce. Therefore, to identify novel BBB targets, we combined transcriptomic analysis of human and murine brain endothelium and performed a complex screening of BBB-enriched genes according to established selection criteria. As a result, we propose the high-affinity cationic amino acid transporter 1 (SLC7A1) as a novel candidate for transport of large molecules across the BBB. Using RNA sequencing and in situ hybridization assays, we demonstrated elevated SLC7A1 gene expression in both human and mouse brain endothelium. Moreover, we confirmed SLC7A1 protein expression in brain vasculature of both young and aged mice. To assess the potential of SLC7A1 as a transporter for larger proteins, we performed internalization and transcytosis studies using a radiolabelled or fluorophore-labelled anti-SLC7A1 antibody. Our results showed that SLC7A1 internalised a SLC7A1-specific antibody in human colorectal carcinoma (HCT116) cells. Moreover, transcytosis studies in both immortalised human brain endothelial (hCMEC/D3) cells and primary mouse brain endothelial cells clearly demonstrated that SLC7A1 effectively transported the SLC7A1-specific antibody from luminal to abluminal side. Therefore, here in this study, we present for the first time the SLC7A1 as a novel candidate for transport of larger molecules across the BBB

Ścieżki sygnalizacyjne ApoE4: wpływ na model bariery krew-mózg in vitro

Bariera krew-mózg (BBB) utrzymuje homeostazę, zapobiegając swobodnemu wnikaniu toksyn i komórek odpornościowych do centralnego układu nerwowego, a także dostarczając składniki odżywcze do mózgu. Zgodnie z obecną hipotezą naczyniowo-mózgową choroby Alzheimera, bariera krew-mózg jest upośledzona już we wczesnych stadiach choroby z powodu zaburzonego usuwania amyloidu beta (Aβ) oraz jego akumulacji w tkance mózgowej. Głównym dowodem na uszkodzenie bariery jest obniżona ekspresja białek złączy ścisłych komórek śródbłonka, a także zwiększona przepuszczalność bariery. Izoformy apolipoproteiny E, głównie E3 i E4, mogą wiązać się z Aβ i wpływać na jego usuwanie. Ponadto gen APOE jest dobrze znanym czynnikiem ryzyka choroby Alzheimera. W niniejszej pracy magisterskiej zbadano wpływ apoE3/4 na ludzkie immortalizowane komórki śródbłonka mózgu ( linia komórkowa hCMEC/D3) stanowiące model in vitro ludzkiej bariery krew-mózg. Za pomocą metody Western blot, zbadano ekspresję dwóch cząsteczek sygnałowych regulujących złącza ścisłe: kinazy białkowej C eta (PKCη) i cyklofiliny A (CypA) po traktowaniu komórek hCMEC/D3 izoformami apoE3/4. Ponadto, aby potwierdzić obecność białek tworzących złącza ścisłe pomiędzy komórkami hCMEC/ D3, przeprowadzono barwienia immunocytochemiczne.Spodziewano się zaobserwować wyższą ekspresję CypA i fosforylowanego PKCη w komórkach traktowanych apoE4 w porównaniu do komórek traktowanych apoE3. Jednakże nie wykazano żadnych znaczących zmian w fosforylacji PKCη i ekspresji CypA. W eksperymentach immunocytochemicznych, po optymalizacjach protokołu zademonstrowano ekspresję białek złączy ścisłych: klaudyny-5 i zona occludens-1 (ZO-1). Komórki linii hCMEC/D3 okazały się odpowiednim modelem dla niniejszego projektu badawczego, niemniej jednak, należy przeprowadzić dalsze badania, aby ocenić czy różne dawki i czas ekspozycji, jak również stan lipidacji izoform apoE mogą przyczynić się do zmian w fosforylacji PKCη jak i ekspresji CypA, a w konsekwencji białek złączy ścisłych.The blood brain barrier (BBB) maintains homeostasis within the brain preventing from free entry of toxins and immune cells as well as providing nutrients into the central nervous system (CNS). According to the recent cerebrovascular hypothesis of Alzheimer's disease (AD), the BBB is compromised in early stages of the disease due to malfunctioned amyloid β (Aβ) clearance and its accumulation within the brain parenchyma. The major evidence for the BBB disruption is decreased expression of endothelial cells tight junction (TJ) proteins and increased permeability of the BBB. Apolipoprotein E isoforms , mostly E3 and E4, may bind to Aβ and affect its clearance. Moreover, APOE gene is a well-known risk factor for AD. Therefore, in the project we tested the effect of apoE3/4 treatment on human immortalized brain endothelial cells (hCMEC/D3) - an in vitro model of human BBB. Expression of two signaling molecules regulating TJ: protein kinase C eta (PKCη) and cyclophilin A (CypA) was examined in Western blot assay after apoE3/4 treatment of hCMEC/D3 cells. Moreover, immunocytochemistry (ICC) studies were performed to confirm the presence of tight junctions between hCMEC/D3 cells.Higher expression of CypA and phosphorylated PKCη in apoE4-treated cells was expected in comparison to apoE3-treated cells. However, no relevant changes in PKCη phosphorylation and CypA expression were shown. Expression of Claudin-5 and zona occludens-1 (ZO-1), two proteins present in TJs was demonstrated in ICC assays after several improvements of the protocol . hCMEC/D3 were proved to be a suitable model for our project, however, further studies need to be performed to assess whether different dosage and exposure time, as well as lipidation status of apoE isoforms may contribute to expression shifts of phosphorylated PKCη, CypA and in consequence, tight junctions proteins

"Application of mesenchymal stem cells in therapies of neurodegenerative diseases"

Zastosowanie mezenchymalnych komórek macierzystych (MKM) jest coraz częściej rozważane jako alternatywna metoda leczenia schorzeń neurodegeneracyjnych, ze względu na ich neuroregeneracyjne i neuroprotekcyjne właściwości. Wykazano, że komórki te silnie oddziałują na układ odpornościowy, co ma kluczowe znaczenie dla obniżenia stanu zapalnego i usuwania złogów źle sfałdowanych białek. Zdolność MKM do różnicowania się w neurony, a także efekty parakrynne wydzielanych przez MKM substancji sprawiają, że obecnie są one intensywnie badane w wielu zwierzęcych modelach chorób neurodegeneracyjnych. W pracy zostały uwzględnione badania przedkliniczne jak i kliniczne mezenchymalnych komórek macierzystych pod kątem zastosowania ich w leczeniu choroby Parkinsona. Porównano efekty działania tych komórek w zależności od źródła ich pozyskiwania i modelu zwierzęcego. W rozdziale 10-tym poruszono temat nowo-poznanej subpopulacji komórek MUSE, która wydaje się posiadać wyjątkowe właściwości regeneracyjne, w tym dla układu nerwowego. Łatwa dostępność autologicznych komórek MKM, brak kontrowersji etycznych wokół ich pozyskiwania i wykorzystanie oraz wysoki potencjał proliferacyjny czynią je doskonałym narzędziem inżynierii tkankowej, w tym zastosowania ich w leczeniu schorzeń centralnego układu nerwowego.Application of mesenchymal stem cells is more and more often considered as an alternative approach to therapy of neurodegenerative diseases in virtue of their neuroregenerative and neuroprotecive properties. It has been demonstrated that these cells have a strong impact on the immune system, which is crucial for the reduction of inflammation and removing deposits of misfolded proteins. The ability of MSCs to differentiate into neurons as well as paracrine effects of MSC’s secretome makes them being extensively studied in numerous animal models of neurodegenerative diseases. In the thesis, both preclinical and clinical trials on MSCs have been discussed in terms of treating Parkinson’s disease. The effects of MSCs depending on the source and the animal model have been compared. In the tenth chapter the subject of the newfound MUSE cells subpopulation has been raised, which seem to have unique therapeutic properties. An easy access of autologous MSCs, no ethical controversy and a high proliferative capacity make them the perfect tissue-engineering tool including application in treatment of the central nervous system disorders

Społeczna odpowiedzialność biznesu na przykładzie L'Oréal Polska

The article aims to assess the benefits of implementing the corporate social responsibility (CRS) concept in enterprises and attempts to examine its impact on society and the environment. The analysis of the effectiveness of investments related to CSR was carried out based on literature review and own research (on-line survey) on the example of L'Oréal Polska. The conclusions from the research confirm the existence of numerous profits related to the implementation of CSR initiatives undertaken by the company. Considering own actions in a forward-looking way and taking responsibility for them shows that CSR is a bridge between the present and the future of business. Therefore, the further development of this concept can be forecasted.Artykuł ma na celu ocenę korzyści płynących z wdrażania koncepcji CRS w przedsiębiorstwach oraz próbę zbadania jej wpływu na społeczeństwo i środowisko. Analiza efektywności inwestycji związanych z CSR została przeprowadzona na podstawie badań literaturowych oraz badań własnych (ankieta on-line) na przykładzie L'Oréal Polska. Wnioski z badań potwierdzają występowanie szeregu profitów związanych z realizacją inicjatyw CSR podejmowanych przez firmę. Patrzenie w sposób przyszłościowy na swoje działania oraz ponoszenie za nie odpowiedzialności wskazuje, że CSR stanowi pomost między teraźniejszością a przyszłością biznesu. Można zatem prognozować dalszy rozwój tej koncepcji

Usage of Converter Gas as a Substitute Fuel for a Tunnel Furnace in Steelworks

Converter gas (BOFG) is a by-product of the steel manufacturing process in steelworks. Its usage as a substitute fuel instead of natural gas for fueling a metallurgical furnace seems to be reasonable due to potential benefits as follows: CO2 emission reduction into the ambient air and savings in purchasing costs of natural gas. Results of theoretical analysis focused on implementing converter gas as a fuel for feeding a tunnel furnace for either steel plate rolling, steel sheet hardening in its real working condition or both, are discussed. The analysis was focused on the combustion chemistry of the converter gas and its potential ecological and economic benefits obtained from converter gas usage to heat up steel in a tunnel furnace. Simulations of combustion were conducted using a skeletal chemical kinetic mechanism by Konnov. The directed relation graph with error propagation aided sensitivity analysis (DRGEPSA) method was used to obtain this skeletal kinetic mechanism. Finally, the model was validated on a real tunnel furnace fueled by natural gas. Regarding exhaust emissions, it was found that nitric oxide (NO) dropped down from 275 to 80 ppm when natural gas was replaced by converter gas. However, carbon dioxide emissions increased more than three times in this case, but there is no possibility of eliminating carbon dioxide from steel manufacturing processes at all. Economic analysis showed savings of 44% in fuel purchase costs when natural gas was replaced by converter gas. Summing up, the potential benefits resulting from substituting natural gas with converter gas led to the conclusion that converter gas is strongly recommended as fuel for a tunnel furnace in the steel manufacturing process. Practical application requires testing gas burners in terms of their efficiency, which should provide the same amount of energy supplied to the furnace when fed with converter gas

Brain endothelial LRP1 maintains blood–brain barrier integrity

Abstract The entry of blood-borne molecules into the brain is restricted by the blood–brain barrier (BBB). Various physical, transport and immune properties tightly regulate molecule movement between the blood and the brain to maintain brain homeostasis. A recent study utilizing a pan-endothelial, constitutive Tie2-Cre showed that paracellular passage of blood proteins into the brain is governed by endocytic and cell signaling protein low-density lipoprotein receptor–related protein 1 (LRP1). Taking advantage of conditional Slco1c1-CreER T2 specific to CNS endothelial cells and choroid plexus epithelial cells we now supplement previous results and show that brain endothelial Lrp1 ablation results in protease-mediated tight junction degradation, P-glycoprotein (P-gp) reduction and a loss of BBB integrity